JP2023530926A - novel food - Google Patents

Abstract

The present invention relates to compositions comprising biomass of thermophilic fungi for use as ingredients in food, beverages, pet food or animal feed. Due to its nutritional composition, thermophilic fungal biomass can be used as a source of protein and/or amino acids, including essential amino acids, lipids, oleic and linolenic acids, dietary fiber, choline and vitamins. Contains vitamins such as E and minerals such as iron. Thermophilic fungal biomass is available as compacted biomass cakes or as a dry powder. Biomass of thermophilic fungi is therefore suitable as an ingredient in meat substitutes and hybrid meat products. In dry powder form, the thermophilic fungal biomass can be easily mixed with other ingredients to produce bakery products such as breads or soups.

Description

Field of Invention

The present invention relates to the field of food supplements and replacements for use with both human food and animal feed.

Background of the Invention

There is an increasing demand for edible products that can provide high protein content from non-animal sources. Driven by increased personal health awareness, edible products containing non-animal source ingredients such as protein and fiber are being considered as healthier alternatives to animal protein-based products. In particular, there is an increasing demand for edible meat substitutes that mimic meat in their composition and texture, but are composed of non-animal ingredients, which reduces reliance on animals such as cattle, such as The carbon footprint of animals can be reduced.

Single-cell protein (SCP) is an interesting alternative to meat protein and is a source of protein in many other food applications such as breakfast cereals, bread, pasta, dairy products, ice cream, chocolate and soups. When fungal SCP is produced, it can be produced from sugar-rich crops in a more sustainable manner compared to meat production, because SCP is produced at a higher rate per hectare than meat production. tonnes of protein and a lower nitrogen footprint. One method of producing a dietary source of protein for human food or animal feed is to produce a “single-cell protein” (SCP) by means of fermentation (Suman et al., 2015, Int J. Curr. Microbiol. Appl. Sci., Vol. 4, No. 9, pp. 251-262). Fermentation in this respect is understood to be the microbial conversion of a carbon-rich feed into a protein-rich product consisting of microbial cells such as bacteria, yeast or fungi. The use of SCP as animal feed and food material offers the additional advantage that the microbial cells have a high content of essential amino acids. Furthermore, fungal cells in particular can be very rich in trace elements and vitamins, making the fermented feed very nutritious.

SCP is already used in food for human consumption. As an example, Quorn™ contains mycoproteins produced as SCPs by fermentation of the fungus Fusarium venenatum, vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 ( niacin), vitamin B5 (pantothenic acid) and biotin (www.mycoprotein.org). Quorn is available in a variety of forms such as sausages, cutlets, burgers, patties and strips, ie meat-like forms.

One issue of SCP production, especially in the case of submerged fermentation with bacteria or yeast, is the concentration of SCP-biomass produced in the fermentation broth. Another problem is the need for expensive enzymes to convert inexpensive polymeric carbon sources into monomeric fermentable sugars. Furthermore, to prevent infection when using mesophilic bacteria for SCP production, it is necessary that sterile fermentation conditions be applied, which leads to prohibitive operating costs due to high capital investment and energy demands. (WO2018/029353). Some of these issues allow the use of non-sterile conditions with submerged fermentation with thermophilic fungi at high temperature and acid pH, and can screen favorable properties of good growth at high temperature and acid pH. It has been investigated by selecting thermophilic fungi combined with morphology and high protein content (WO2018/029353).

However, there remains a need for non-animal protein sources that offer a wider range of uses in addition to the meat-like forms currently available.

[Summary of Invention]
In a first aspect, the present invention provides a thermophilic thermophile as a source of at least one of a) proteins and/or amino acids, b) lipids, c) dietary fiber, d) choline and/or vitamins and e) minerals. The use of compositions comprising fungal biomass in at least one of food, beverage, pet food and feed products.

In a preferred use according to the present invention, the composition containing thermophilic fungal biomass is prepared by a) growing a thermophilic fungal strain in submerged culture in a medium and b) sieving, filtering and decanting. recovering the fungal biomass from the medium by at least one of and c) optionally further drying the biomass cake by pressing out remaining water to obtain a biomass cake having a dry matter concentration of at least 20%. or a composition comprising biomass of thermophilic fungi obtained in claim 2, or by flash-drying, pulverizing the biomass cake obtained in claim 2, by warm air, preferably by freeze-drying under vacuum , preferably a dry powder obtainable by further drying to a biomass powder to a moisture content of 7 (w/w)% or less.

In a preferred use according to the invention the fungal strain is Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor (Rhizomucor ), Rhizopus, Thermomyces, Myceliophthora, Thermoascus, Thielavia, Mucor, Stibella, Melanocarpus, Malblanchea ( Malbranchea), a strain of the fungal genus selected from the group consisting of Dactylomyces, Canariomyces, Scytalidium, Myriococcum, Corynascus and Coonemeria, more preferably the genus is Rhizomucor, more preferably the strain is Rhizomucor pusillus, most preferably the strain is Rhizomucor pusill CBS143028, or a single colony isolate or derivative thereof.

In a preferred use according to the invention at least one of the proteins and amino acids is aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine , histidine, arginine, cysteine, methionine and tryptophan, preferably the amino acids are essential amino acids, more preferably threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine and It contains essential amino acids selected from the group consisting of tryptophan, most preferably in vegetarian and vegan foods. In a more preferred use according to the invention a) the biomass of thermophilic fungi is used as a (dietary) source of at least one of proteins, amino acids and essential amino acids in vegetarian or vegan foodstuffs, b) thermophilic The biomass of the fungus is used as a (dietary) source citrulline in food, food supplements or beverages for sports enthusiasts or c) the biomass of the thermophilic fungus is used to produce proteins, amino acids and essential amino acids in pet food. preferably as a replacement for standard animal or vegetable sources of protein, amino acids in pet food, more preferably as the sole source of protein and/or amino acids .

In a preferred use according to the invention, the thermophilic fungal biomass is made from i) meat substitutes, ii) hybrid foods containing meat and non-meat materials, iii) foods rich in carbohydrates, iv) refined flours. and v) pet food, preferably the dietary fiber comprises chitosan. In a more preferred use according to the invention, the thermophilic fungal biomass is used as a dietary fiber source in food for humans or pets, preferably to provide satiety to humans or pets in need of weight management or weight loss. and preferably the dietary fiber comprises chitosan.

In a preferred use according to the invention, thermophilic fungal biomass is used as a choline source in vegan or vegetarian food or in pet food, preferably cat or dog food.

In a preferred use according to the invention the biomass of a thermophilic fungus is used as a source of at least one of retinol, vitamin E, riboflavin, pyridoxine and folic acid, preferably said biomass contains retinol in cat food. used as a source.

In a preferred use according to the invention, thermophilic fungal biomass is used as iron source, preferably thermophilic fungal biomass is used to replace soybean as iron source, more preferably thermophilic fungal biomass. Biomass is used to replace soybeans as an iron source in meat substitutes.

In a preferred use according to the invention the biomass of a thermophilic fungus is used as a source of at least one of the monounsaturated and polyunsaturated fatty acids, preferably the biomass thermophilic fungus contains oleic acid and linoleic acid used as a source of at least one of

In a preferred use according to the present invention, thermophilic fungal biomass is used in food, beverage, pet food and feed products, either in cake or powder form, at a maximum level of 70 (w/w) and at least 1 Used to be incorporated at a minimum level of (w/w)%.
In a preferred use according to the invention, the thermophilic fungal biomass is a) a meat substitute and not more than 42 (w/w) % thermophilic fungal biomass in cake form, or 14 (w/w) % or less of biomass of thermophilic fungi in powder form, b) a hybrid meat product, wherein no more than 52% (w/w) of biomass of thermophilic fungi in cake form, or Hybrid meat products containing no more than 17.3% (w/w)% biomass of thermophilic fungi in powder form, c) cereal-based foods, no more than 30% (w/w) in cake form or a cereal-based food product comprising no more than 10 (w/w)% of the thermophilic fungal biomass in powder form, d) a ready-to-eat dietary food product comprising 16 ( w/w)% of thermophilic fungal biomass in cake form, or 5.3% (w/w)% or less of thermophilic fungal biomass in powder form, e) Ready-to-eat soups containing no more than 27 (w/w)% thermophilic fungal biomass in cake form or no more than 9 (w/w)% thermophilic fungal biomass in powder form. and f) a food for sports enthusiasts, comprising no more than 70% (w/w) biomass of thermophilic fungi in cake form or in powder form. used as an ingredient for food in

In a second aspect, the present invention provides a food product comprising a composition comprising biomass of a thermophilic fungus as defined herein, a) a meat substitute, up to 42 (w/w)% of biomass of thermophilic fungi in cake form, or 14 (w/w)% or less biomass of thermophilic fungi in powder form, b) hybrid meat products, 52 (w /w) % or less thermophilic fungal biomass in cake form, or 17.3 (w/w)% or less of thermophilic fungal biomass in powder form, c) a cereal base cereal base containing no more than 30% (w/w)% biomass of thermophilic fungi in cake form or no more than 10% (w/w)% of biomass of thermophilic fungi in powder form d) a ready-to-eat dietary food containing no more than 16 (w/w)% of thermophilic fungal biomass in cake form or no more than 5.3 (w/w)% in powder form A ready-to-eat dietary food product comprising a thermophilic fungal biomass, e) a ready-to-eat soup and not more than 27 (w/w) % thermophilic fungal biomass in cake form, or 9 (w/w) w) % or less ready-to-eat soup containing biomass of thermophilic fungi in powder form, and f) food for sports enthusiasts, no more than 70 (w/w) % in cake form or powder. It relates to a food product, which is a food product for sports enthusiasts, containing biomass of thermophilic fungi in the form.

[Description of the Invention]
definition
definition
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. In fact, the invention is by no means limited to this method.

For purposes of the present invention, the following terms are defined below.

As used in the present invention, the term "and/or" means that one or more of the recited instances may be used alone or from at least one of the recited instances to all of the recited instances. to indicate what can happen.

As used in the present invention, a particular value followed by "at least" means that particular value or more. For example, "at least two" is understood to be the same as "two or more", i.e., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc. be done.

When the word "about" or "approximately" is used in connection with a numerical value (e.g., about 10), preferably the value is greater than or less than 0.1% of the value given (10 ) value.

The term "unicellular protein" is abbreviated as "SCP" and is used herein from cells of organisms existing in a unicellular or unicellular state, including unicellular bacteria, yeast, fungi or algae. This biomass, understood to refer to the biomass of which it consists essentially, preferably in dry form, is suitable as a dietary source of protein or protein supplements in human food or animal feed.

A "fungus" is defined herein to be a eukaryotic microorganism and includes all species of the subdivision Eumycotina (Alexopoulos et al., 1962, in Introductory Mycology, John Wile & Sons, Inc., New York). . The term "fungi" thus includes both filamentous fungi and yeasts. "Filamentous fungi" are defined herein to be eukaryotic microorganisms that include all filamentous forms of the subdivisions Eumicotina and Oomycota (Hawksworth et al., Ainsworth and Brisby's Dictionary of the Fungi, vol. 7th edition, Commonwealth Mycological Institute, Kew, Surrey, 1983). Filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, and carbon metabolism is strictly aerobic. Thermophilic fungi for use in the present invention are fungi that grow at temperatures of at least 45°C and sometimes even above 56°C.

The term "fermentation" or "fermentation process" is broadly defined herein according to its general definition as used in industry as any (large-scale) microbial process that occurs in the presence or absence of oxygen, It comprises culturing at least one microorganism whereby preferably the microorganism produces a useful product at the expense of consuming one or more organic substrates. Therefore, the term "fermentation" is used herein rather than a rigorous, scientific definition as it is defined as a defined microbial process in which microorganisms extract energy from carbohydrates in the absence of oxygen. It has a much broader definition. Similarly, the term "fermented product" is broadly defined herein to be any useful product produced in a (large scale) microbial process occurring in the presence or absence of oxygen.

The term "TOTOX value", an abbreviation for total oxidation value, is understood to indicate the oxidative load to which the fat is exposed. The TOTOX value is a scaleless number used to describe the total oxidative load to which fat is exposed. This value is calculated as the sum of the anisidine value and twice the peroxide value. Anisidine value (AnV) is a measure of aldehyde formation during fat oxidation. This value represents the absorption of fat in response to p-anisidine (the trivial name for p-methoxyaniline) under specified conditions. It is used to characterize the oxidation history of fats because aldehydes usually originate from the oxidation of unsaturated fatty acids. Peroxide value (PV) is a measure of the degree of oxidation of a fat or oil. This value indicates the quality of the oxidant, usually a hydroperoxide, that releases iodine from potassium iodide under certain conditions. PV is expressed in milliequivalents of reactive oxygen species per kg of fat (Beare-Rogers et al., Lexicon of of lipid nutrition (IUPAC Technical Report), 2009, DOI: 10.1351/pac200173040685). Oils with a TOTOX value of less than 80 were described as having good aroma ratings (JP2014054248).

Detailed description of the invention

The inventors have found that biomass derived from thermophilic filamentous fungi, such as Rhizomucor pusillus strains, produced through a fermentation process during which simple carbohydrates and non-organic nitrogen sources are converted into highly nutritious biomass is compacted. (referred to as a "cake") and finally dried to obtain a powder. The fungal biomass thus obtained can be obtained from its source, eg Rh. Psillus is considered a novel food within the European Union (EU) if it has no history of human consumption within the EU. Fungal biomass, for example in cake form and powder form, can be used as an ingredient in various food categories at different levels of use. Thanks to its texture, the fungal biomass of the present invention is a versatile material for many food products. Fungal biomass in cake form represents a valuable material for meat substitutes and hybrid meat products. Fungal biomass in its powder form can be easily mixed with other ingredients to produce bakery products such as bread or soups due to its powder-like texture. Also, the fungal biomass of the present invention can be used as a feed material, which is an alternative to or improves the nutritional value of feeds of animal and vegetable origin.

Additionally, the inventors have found that the fungal biomass of the present invention contains a number of valuable nutritional components and thus can be used as a dietary source for these components.

In a first aspect, the present invention therefore provides in one of food, beverage, pet food and feed products a) proteins and/or amino acids, b) lipids, c) dietary fiber, d) choline. and/or the use of a composition comprising thermophilic fungal biomass as a source of at least one of vitamins and e) minerals. Preferably, the composition contains a) proteins and/or amino acids, b) lipids, c) dietary fiber, d) choline and/or vitamins in one of food, beverage, pet food and feed products. and e) used as a dietary source of at least one of the minerals. The term "dietary source of," as used herein, refers to the consumption of products that contain a particular dietary ingredient.

A composition comprising thermophilic fungal biomass for use according to the invention is preferably or can be obtained in one of the following methods.

In one embodiment, the composition comprising thermophilic fungal biomass is prepared by: a) growing a strain of thermophilic fungus in submerged culture in a medium; and b) at least one of sieving, filtering and decanting. recovering the fungal biomass from the medium to produce a biomass cake by preferably sieving, filtering or decanting the biomass cake with a dry matter concentration of at least 12% (w/v) and c) optionally further drying the biomass cake by pressing out remaining water to obtain a biomass cake having a dry matter concentration of at least 20%. Or the resulting cake.

In another embodiment, the composition comprising thermophilic fungal biomass is obtained by grinding the biomass cake obtained in b) or c) above, by warm air, by freeze-drying, preferably under vacuum, or by flashing. Dry powder obtained or obtainable in the above-defined method, further comprising step d) further drying to a biomass powder, preferably to a moisture content of 7% (w/w) or less. More preferably, the water content is 5 (w/w)% or less.

Methods, conditions and media for growing thermophilic fungi in submerged culture in media are described in detail in WO2018/029353 or US2019174809, which are incorporated herein by reference. Preferably, the method comprises the steps of a) growing the thermophilic fungus in a medium containing a feed rich in fermentable carbon, a nitrogen source and additional components necessary for the growth of the fungus. Preferably, in step a) the fungus is grown in submerged culture. Preferably, in step a) the fungus is grown under non-sterile conditions. Preferably, in step a) the fungus is , 50, 51, 52, 53, 54 or 55° C. or higher. Preferably, in step a) the fungus is , 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2 Grow at a pH of less than .8, 2.7, 2.6 or 2.5.

In one embodiment of the method of the invention, the thermophilic fungal strain is preferably grown or cultured in a chemically defined medium. The term "chemically defined" refers to a fermentation medium consisting essentially of chemically defined constituents, i.e., a fermentation medium in which the chemical composition of essentially all chemicals used in the medium is known. It is understood to be used for culture medium.

Therefore, in one embodiment, the thermophilic fungal strain is cultivated in a chemically defined medium consisting of a carbon source, a nitrogen source, and additional components necessary for fungal growth.

The carbon source preferably comprises or consists of at least one of carbohydrates and organic acids. Preferably, the carbohydrate comprises a source of at least one of glucose, fructose, galactose, xylose, arabinose, rhamnose, fucose, galactose and mannose, of which glucose and fructose are preferred and glucose is most preferred. . Suitable carbohydrate carbon sources, including, for example, sources of glucose and/or fructose, include, for example, maltose, isomaltose, maltodextrin, starch, syrups of glucose (e.g. corn syrup such as HCFS), inverts (sucrose or sugar beet) sucrose, crude starch, liquefied starch (eg by liquefaction using alpha amylase, e.g. Liquozyme (Novozymes) or Veretase (BASF), inulin, raffinose, melibiose and stachyose. Included in the carbon source) Organic acids that may be used include lactic acid, acetic acid, galacturonic acid, gluconic acid.

Nitrogen sources in chemically defined media to be used in the methods of the invention include uric acid, ammonia, nitrate, ammonium salts such as ammonium sulfate, ammonium phosphate and ammonium nitrate, and amino acids such as glutamate and lysine. preferably comprises or consists of at least one of More preferably, the nitrogen source is selected from the group consisting of ammonia, ammonium sulfate and ammonium phosphate. Most preferably, the nitrogen source is ammonia.

Further components necessary for fungal growth include catalytic elements, eg components of enzymes or enzyme cofactors. These elements include, for example, magnesium, iron, copper, calcium, manganese, zinc, cobalt, molybdenum, selenium and borium. In addition to the structural catalytic elements mentioned above, cations such as potassium and/or sodium are preferably present to serve as counterions and control intracellular pH and osmolarity. Suitable mineral compositions for chemically defined media of the present invention are described in US Pat. No. 20140342396A1, incorporated herein by reference.

Compounds that may optionally be included in the (chemically defined) medium for growing the fungi are chelating agents such as citric acid, and buffering agents such as monopotassium phosphate and dipotassium phosphate, calcium carbonate etc., as well as vitamins. Buffers are preferably only added when working with methods without external pH control. Vitamins refer to a group of structurally unrelated organic compounds that may be required for normal metabolism of thermophilic fungi. Fungi are known to vary widely in their ability or inability to synthesize the vitamins they require. Vitamins only need to be added to the fermentation medium of fungal strains incapable of synthesizing said vitamins. Typically, chemically defined fermentation media for lower fungi, such as Mucorales, may require supplementation with one or more vitamin(s). Higher fungi often do not have vitamin requirements. Vitamins are selected from the group consisting of thiamine, riboflavin, pyridoxal, nicotinic acid or nicotinamide, pantothenic acid, cyanocobalamin, folic acid, biotin, lipoic acid, purines, pyrimidines, inositol, choline and hemin. In a preferred embodiment, however, the thermophilic fungi grown in the method of the invention are strains that do not require the presence of any vitamins in the chemically defined medium. In fact, the inventors have found that species of the genus Rhizomucor, such as strains Rhizomucor pcilus CBS 143028, Rhizomucor miehei CBS 143029 and Rhizopus sp. CBS 143160, do not require any vitamins, even when grown on mineral media. I found out.

In a further preferred embodiment, the fungus is grown in step a) in the medium defined herein in the presence of an antifoaming agent. Defoamers are generally well known in the art (see, eg, https://en.wikipedia.org/wiki/Defoamer). Antifoam agents are chemical additives which reduce and prevent foam formation in industrial process liquids, for example fermentation broths. Strictly speaking, anti-foam agents remove existing foam and anti-foam agents prevent further foam formation, although the terms "anti-foam agent", "anti-foam agent" and "anti-foam agent" are used. "Foam agent" is used interchangeably herein. Antifoaming agents suitable for use in the fermentation process of the present invention are preferably at least one of an oil-based antifoaming agent, a polyalkylene glycol-based antifoaming agent and a silicon-based antifoaming agent. Examples thereof include vegetable or mineral oils, respectively, and animal fats, polypropylene glycol (PPG) or polyethylene glycol (PEG) and Antifoam C100K (Basildon Chem. Comp. Ltd., Abingdon, Oxford, England). The antifoaming agent is preferably a food grade antifoaming agent. A preferred food grade antifoam agent is a clean label antifoam agent. A preferred antifoaming agent for use in the method of the present invention comprises or consists of a vegetable oil, preferably an edible vegetable oil. Preferred (edible) vegetable oils for use as antifoaming agents in the process of the invention are canola (rapeseed) oil, coconut oil, corn oil, cottonseed oil, olive oil, palm oil, palm kernel oil, linseed oil, peanut oil. , safflower oil, soybean oil, sunflower oil and high oleic sunflower oil.

In one embodiment, the thermophilic fungi grown to produce the composition comprising thermophilic fungal biomass according to the invention are preferably filamentous fungi. Preferred thermophilic fungi for use in the present invention are Rasamsonia, Taralomyces, Penicillium, Acremonium, Humicola, Paechylomyces, Chaetomium, Rhizomucor, Rhizopus, Thermomyces, Micheliohutra, Thermoascus, Tierawia, Mucor, Tibera, Melanocarpus, Malblanchea , Dactylomyces, Canaryomyces, Scytharidium, Myriococcum, Corynascus and Coronemeria. More preferably, the thermophilic fungus is Rasamsonia composticola, Talaromyces emersonii, Rhizomucor miehei, Rhizomucor pusillus, Thermomucor indica-seudat icae), Tierrawia A strain of a fungal species selected from the group consisting of Thielavia terricola, Thielavia terrestris, Thermoascus thermophilus, among which the strain Rasamsonia compoposticola CBS 141695 ( Thermomucor indicae-ceudaticae CBS 143027 (deposited July 21, 2017), Rhizomucor miehei CBS 143029 (deposited July 21, 2017), Rhizomucor pcilus CBS 143028 (deposited July 21, 2017) Deposited July 21), Thermoascus thermophilus CBS 528.71 and Tierawia terrestris CBS 546.86 are preferred.

In preferred embodiments, the thermophilic fungus is a strain of the class Zygomycete, of which the family Mucoraceae is preferred. More preferably, the thermophilic fungus is a strain of a genus selected from the genera Mucor, Rhizomucor and Rhizopus, of which the genus Rhizomucor is preferred. Most preferably, the thermophilic fungus is of the species R. endophyticus, R. miehei, R. pakistanicus, R. tauricus, R. walliabilis (R. variabilis) and R. pusillus, among which the species Rhizomucor pusillus is preferred. Preferred strains of the aforementioned thermophilic fungi for use in the present invention include the Westerdijk Institute for Fungal Diversity, Uppsalaan 8, 3584 CT Utrecht, The Netherlands (formerly Centraalbureau voor Schimmelcultures, CBS, under the rules of the Budapest Treaty). and Rhizomucor meihei CBS 143029 and Rhizopus sp. CBS 143160 (deposited on Aug. 11, 2017), of which Rhizomucor pusillus CBS 143028 is most preferred.

In one embodiment the method of the invention is a batch method, more preferably at least step a) of the method is performed as a batch method. In a preferred embodiment, however, the method of the invention, or preferably at least step a) of the method, is a fed-batch method, a repeated fed-batch method (where iteratively a portion of the fermentation broth is harvested) or It is carried out in such a way that it is a continuous process. In one embodiment the method in step a) is a carbon limited method, preferably the carbon source is supplied at a growth limiting rate by continuous or intermittent feeding.

In one embodiment, following fermentation, the fungal biomass may be pasteurized, preferably at a temperature of at least 60° C., as described in co-pending application EP20161554.9. Preferably, the biomass is pasteurized at a temperature of at least 70°C for up to 45 minutes. In a preferred embodiment, pasteurization is carried out in an in-line heating unit preferably comprising pipe heaters, heating blocks or steam infusion elements, more preferably steam infusion elements, wherein the in-line heating unit is optionally with a mixing element such as a static mixer. Preferably, pasteurization is carried out for up to 5 minutes, preferably from about 0.5 to about 3 minutes, more preferably from about 1 to 2 minutes. Preferably, pasteurization is carried out at a temperature of at least 74°C, preferably at least 80°C, more preferably at least 86°C. Preferably, the microbial count of the pasteurized biomass has a log ₁₀ reduction of at least 7, preferably of at least 11, more preferably of at least 12 compared to the supplied biomass prior to pasteurization. In a preferred embodiment, the biomass flows through an in-line heating unit having a residence time of about 1-2 minutes at a temperature of about 86° C., where the pH of the biomass is preferably up to 4.5 and more than Preferably it is at most 3.5.

In one embodiment, antioxidants are added to the biomass. Preferably, antioxidants are added prior to pasteurization of the biomass, as described, for example, in co-pending application EP20161606.7. Preferably, the antioxidant is a free radical scavenger such as carnosol; carnosic acid; rosemary extract; flavan-3-ols such as epicatechin, epigallocatechin, epigallocatechin gallate (EGCG), epigallocatechin gallate (EGCG); catechins; flavonoids such as kaempferol, quercetin or myricetin; green tea extract; butylated antioxidants such as butylated hydroxytoluene (BHT), butylated hydroxyanisole or tert-butylhydroquinone (TBHQ); gallic acid; caffeic acid esters, such as methyl caffeate, ethyl caffeate, caffeic acid phenethyl ester (CAPE) or rosmarinic acid; spearmint extract; vitamin E, such as tocopherols or tocotrienols; , such as retinol, retinoleic acid or retinal; provitamin A such as carotenoids, beta-carotene, lutein, zeaxanthin or echinenone; quinolone antioxidants such as ethoxyquin; or seaweed extracts; or oxygen reducing agents such as ascorbic acid or acerola. an extract; or at least one of a chelating agent such as citric acid or EDTA. Preferably, the resulting biomass composition has a TOTOX value of less than 50, preferably less than 35, more preferably less than 20, wherein the TOTOX value is preferably less than 50 for at least 6 weeks, More preferably it remains below 40.

The fungal biomass is then preferably separated from the fermentation medium. Therefore, in one embodiment, a method of preparing a composition comprising a thermophilic fungal biomass grown in (step a) from the culture medium, preferably by b) at least one of sieving, filtering and decanting ) further comprising the step of fungal biomass recovery. More preferably, the biomass is recovered from the medium by at least one of rotary drum filtration, filter press, belt filter, decanter centrifugation and sieving. Preferably, the biomass has preferably 100, 200, 300, 400 or 500 μm or a pore diameter of 0.6, 0.7, 0.8, 0.9, 1.0, 1.5 or 2 mm recovered by sieving over a sieve or screen having a The biomass can be collected on a sieve or screen by at least 2, 3 or 4 rounds of sieving in succession, whereby smaller diameter pores are applied in each subsequent round of sieving. For example, a first round of sieves with a hole diameter of 2 mm, subsequent rounds of 1, 0.5 and/or 0.1 mm. Optionally, the dry matter concentration of the screened, filtered or decanted biomass (cake) is further increased by further removal of water, i.e. drying. The biomass cake is further extruded, for example, by pressing out (most of) the remaining water using compressed air, for example, using pneumatic pressure, and/or mechanical pressing, for example, using a belt press or a screw press. can be dried. The biomass cake thus obtained preferably has a dry matter concentration of at least 12, 15, 20, 25, 30, 35, 40, 45 or 5 (w/w) %.

In another embodiment, the biomass can be pressed into a cake and optionally the cake can be crushed or extruded to allow for example drying, preferably air drying. Preferably, the particle size of the pressed mycelial biomass cake is reduced by physical means to allow (more efficient) drying of the pressed cake. This is optionally 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4 using extruders known per se in the art. , 1.6, 1.8 or 2 mm diameter by extrusion of the mycelium cake. Alternatively, the cake particle size can be reduced by a combination of grinding and sieving. As a grinding step any type of mill known per se in the art, such as a knife mill or a hammer mill, can be used. To obtain a homogeneous particle size of the pressed and ground cake, the larger particles still present after grinding are 0.5, 1.0, 1.2, 1.4, 1.6, 1.8, It can be removed before drying by sieving with a pore diameter size of 2.0, 2.5 or 3 mm sieve. The resulting milled cake will preferably have a particle size of between 1 mm and 3 mm before drying. Drying of the extruded or ground cake is preferably carried out at a temperature of 30-70°C. Hot air then gently and cost effectively dries the cake in a belt dryer or fluid bed dryer. Steam drying at high temperatures (e.g. >80°C) is preferably avoided because it negatively affects the digestibility of proteins through Maillard reactions, through denaturation and baking of amino acids and also through chemical degradation. This is because there is a risk that Alternatively, the extruded or milled powder is dried under vacuum in a freeze drying process or by flash drying. The biomass in the form of a dry powder thus obtained preferably has a moisture content of 5, 4, 3, 2 or 1 (w/w) % or less.

The cake form of thermophilic fungal biomass may be frozen and stored at -18°C, while the powder form may be stored at room temperature.

Proteins and Amino Acids In one embodiment, the composition comprising biomass of the thermophilic fungi of the invention is used as a (dietary) source of protein. Preferably, the protein consists of aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, methionine and tryptophan. Amino acids selected from the group.

In one embodiment, the composition comprising thermophilic fungal biomass of the invention is used as a (dietary) source of amino acids. In preferred embodiments, the amino acids are aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, methionine and tryptophan. is an amino acid selected from the group consisting of

Protein represents the major source of nitrogen and is an essential amino acid in the human diet. Both are fundamental for tissue growth and maintenance [3]. The main sources of protein in the human diet are of animal origin (meat, fish, eggs, dairy products) or of vegetable origin, eg legumes, nuts, cereal products. However, proteins of animal origin are among the highest quality in terms of amino acid composition. All amino acids are important in human nutrition, but some cannot be produced de novo by humans and therefore can only be introduced through the diet. These amino acids are defined as essential amino acids. Amino acids that are essential for humans are threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine and tryptophan [3].

In one embodiment, the composition comprising thermophilic fungal biomass of the present invention is used as a (dietary) source of protein containing essential amino acids. Preferably, the essential amino acids are selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine and tryptophan.

In one embodiment, the composition comprising biomass of thermophilic fungi of the invention is used as a (dietary) source of essential amino acids. Preferably, the essential amino acids are selected from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine and tryptophan. More preferably, the composition comprising the thermophilic fungal biomass of the invention is used as a (dietary) source of all essential amino acids.

Citrulline malate is available and widely used as a product by sports enthusiasts to improve athletic performance and muscle recovery. Supplementation with L-citrulline has been shown to improve vascular function, thus improving exercise performance and reducing muscle fatigue after exercise [4]. Besides its high protein content and the presence of all essential amino acids, the fungal biomass of the invention represents a good material for food for sports enthusiasts due to its high citrulline content. In one embodiment, the composition comprising biomass from thermophilic fungi is used as a (dietary) source of citrulline. Preferably, the composition is used in a sports enthusiast's food, food supplement or beverage.

The high protein content of the fungal biomass of the present invention makes it a good substitute for animal protein in vegetarian and vegan foods. In one embodiment, the biomass from thermophilic fungi is therefore used as a (dietary) source of at least one of the herein defined proteins, amino acids and essential amino acids in vegetarian or vegan food products. .

Fiber In one embodiment, the composition comprising the thermophilic fungal biomass of the present invention is used as a (dietary) source of dietary fiber.

Dietary fiber is defined as non-digestible carbohydrates that play an important role in the function of the human intestine. The Adequate Intake (Adequate Intake, AI) of dietary fiber for adults (both genders) for normal bowel movements was set at 25 g/day according to the EFSA DRV. Dietary fiber intakes higher than 25 g/day in adults have been shown to reduce the risk of coronary artery disease, the risk of type 2 diabetes and aid weight maintenance. Furthermore, a daily intake of high fiber has been shown to be associated with a lower risk of colorectal cancer [1]. Dietary fiber is fermented by bacteria that live in the large intestine, which play an important role in the microbial composition of the intestinal flora. Microbial fermentation of dietary fiber can supply many other micronutrients, such as vitamins and short-chain fatty acids (SCFAs), which can be absorbed by the human intestine [2]. The major sources of dietary fiber in the human diet are whole grain cereals, vegetables, fruits and potatoes.

The biomass of the thermophilic fungi of the invention has a relatively high content of dietary fiber (10.6 g dietary fiber/100 g or 37% dry matter). The fungal biomass of the present invention is therefore an excellent source of dietary fiber for use in food, beverage, pet food or feed products.

In particular, a high dietary fiber content allows dietary fiber to be added to foods that are generally poor in fiber. One example of this is meat substitutes made with the fungal biomass of the present invention, wherein the proteins of the thermophilic fungal-derived biomass can replace the animal protein contained in the meat product while simultaneously However, meat substitutes are enriched with dietary fiber, which is essentially absent from meat products.

In one embodiment, the thermophilic fungal-derived biomass of the invention is therefore used as a source of dietary fiber in food products that are meat substitutes or hybrid meat products. Due to its texture, the cake form of biomass derived from thermophilic fungi is particularly suitable as an ingredient for meat substitutes and hybrid meat products.

Similarly, dietary fiber and protein are essential micronutrients in pet food. The primary companion animals, dogs and cats, have different requirements for dietary fiber and protein. Furthermore, the essential amino acids required in a pet's diet differ from those required in a human diet. Currently, protein sources for pet food are represented by materials of animal origin (poultry, bovine, porcine, fish, eggs) or vegetable materials such as corn, rice, beans and soybeans. In addition, dietary fiber is important for stool quality, weight control and satiety in pets and humans [5].

In one embodiment, therefore, the thermophilic fungal-derived biomass of the present invention is used as a source of dietary fiber to enrich the fiber content of food products. Foods to be enriched with dietary fiber are i) meat substitutes, ii) hybrid foods containing meat and non-meat ingredients, iii) foods rich in carbohydrates, iv) made with refined flours. and v) pet food. More particularly, the foods to be enriched with dietary fiber are pizza and pizza-like dishes, savory pies and tarts, ready-to-eat soups, pasta and similar products, premixes for baking products (dry), biscuits, Cakes, pancakes, breads and rolls, pizza bases (prepared), gluten-free breads, porridge (dry/ready to eat), cereal bars, cereal flakes and similar, muesli and mixed breakfast cereals, for sports enthusiasts It can be a food product selected from food, food for weight loss, dairy ice cream and the like, fermented milk products, chocolate and chocolate products.

In one embodiment, therefore, the thermophilic fungal-derived biomass of the present invention is preferably used as a source of dietary fiber in human or pet food to provide satiety to humans or pets in need of weight management or weight loss. used as

Micronutrients The thermophilic fungal-derived biomass of the present invention has a high content of choline (Table 8). Although choline can be synthesized de novo by the human body, choline still represents a fundamental component of the human diet because the amount de novo generated is 400 mg/day for adults (both genders). This is because it is not always sufficient for an adequate intake [9]. In contrast, choline is an essential vitamin for dogs and cats. For adult cats, the minimum nutritional level of choline required is 60 mg/kg metabolic body weight (BW), while for adult dogs it is 45 mg/kg metabolic BW [11]. Therefore, the thermophilic fungal-derived biomass of the present invention is a good source of choline for human and animal nutrition, e.g. can be used as a choline source.

In one embodiment, the thermophilic fungal-derived biomass of the present invention is used as a (dietary) source of choline in vegan or vegetarian food or in pet food, preferably cat or dog food.

In addition to choline, the thermophilic fungal biomass of the present invention contains other vitamins that are essential for humans and/or animals, such as retinol (vitamin A), vitamin E, riboflavin (vitamin B2), pyridoxine (vitamin B6) and Contains folic acid (vitamin B9/B11) (see Table 8).

Vitamin E is known to have antioxidant activity [13]. Vitamin B2, also known as riboflavin, is involved in diverse reactions in the human body [14], while vitamin B6 is involved in different processes such as amino acid metabolism, one-carbon metabolism, glycogenolysis and gluconeogenesis and heme synthesis. It plays an important role in metabolic pathways [15].

In one embodiment, therefore, the thermophilic fungal-derived biomass of the present invention is a (dietary) source of at least one of retinol (vitamin A), vitamin E, riboflavin (vitamin B2), pyridoxine (vitamin B6) and folic acid. used as

Vitamin A plays an important role in the mechanism of phototransduction of the eye in both humans and animals [12]. Vitamin A, in particular, is an essential vitamin for cats [11]. Therefore, in a preferred embodiment, the thermophilic fungal-derived biomass of the present invention is used as a (dietary) source of retinol in cat food.

Minerals The thermophilic fungal-derived biomass of the present invention has a high content of different minerals (Table 10). This is due to minerals added during the fermentation process and required for fungal growth. This means that the mineral composition of Rhizomucor pusillus biomass can be modified and/or standardized based on its use in different food or feed products.

In one embodiment, the composition comprising thermophilic fungal biomass of the present invention is used as a (dietary) source of minerals selected from the group consisting of calcium, magnesium, phosphorus, potassium, zinc, iron, copper and manganese. be.

The iron (Fe) content in the fungal biomass of the present invention was particularly high and represented a limiting factor for uptake assessment according to EFSA DRV. The dose of iron used in the fermentation of the fungal biomass of the invention had to be further reduced to reach a final concentration of 14 mg/kg (Table 10).

The thermophilic fungal-derived biomass of the present invention can therefore be considered a valuable iron source in replacing other plant products, such as soybeans, from which meat substitutes are made (see Table 11). Soy products are commonly introduced into vegetarian or vegan diets due to their high iron content. However, soy cannot be introduced into everyone's diet in the event of an allergy. Soybean is actually considered to be one of the major food allergens. The thermophilic fungal-derived biomass of the present invention can be used to replace soybeans as an iron source in vegetarian and vegan diets. Therefore, in one embodiment, thermophilic fungal biomass is used as an iron source. In a preferred embodiment, thermophilic fungal biomass is used to replace the iron source. In a more preferred embodiment, the thermophilic fungal biomass is used to replace soybean as an iron source, more preferably the thermophilic fungal biomass is used to replace soybean as an iron source in meat substitutes. used.

Lipids The thermophilic fungal-derived biomass of the present invention contains, on a dry matter basis, about 8% (w/w) fat, of which about 16-22% (w/w) consists of saturated fatty acids, and about 50-56% (w/w) of which consists of saturated fatty acids. % w/w) consists of monounsaturated fatty acids (mostly oleic acid) and about 27-28% (w/w) consists of polyunsaturated fatty acids (mostly linoleic acid). In one embodiment, therefore, biomass thermophilic fungi are used as a source of at least one of monounsaturated and polyunsaturated fatty acids. More preferably, biomass thermophilic fungi are used as a source of at least one of oleic acid and linoleic acid.

Food, Beverage, Pet Food and Animal Feed In a further aspect, the present invention relates to food, beverage, pet food and feed products comprising the thermophilic fungal-derived biomass of the present invention.

The biomass of the thermophilic fungi of the present invention in either cake or powder as described herein can be used as ingredients in different food categories with different levels of use. The maximum use level of the thermophilic fungus of the invention in different food categories (Table 1) is determined by the European Food Safety Authority (EFSA) dietary intake for macronutrients and micronutrients contained in the thermophilic fungus of the invention. Based on the reference (DRV), furthermore on the background diet-based intake assessment of different European populations have been set.

In one embodiment, the thermophilic fungus-derived biomass is added into at least one of the foods and beverages according to the European Food Safety Authority (EFSA) Dietary Reference Values (DRV) for thermophilic fungi-derived biomass. is incorporated at a level less than or equal to the maximum use level of

Therefore, biomass derived from thermophilic fungi can be used in food, beverage, pet food and feed products in either cake or powder form 70, 65, 60, 55, 52, 50, 45, 42, 40, It is used for incorporation at maximum levels of 35, 30, 25 or 20 (w/w)%. Preferably, the food, beverage, pet food and feed products comprise at least 1, 2, 5, 10 or 15 (w/w)% thermophilic fungus-derived biomass in either cake or powder form. .

In one embodiment, the thermophilic fungus-derived biomass is used as material for a meat substitute, wherein preferably the meat substitute contains no more than 42 (w/w) % thermophilic fungus in cake form. of biomass, or up to 14% (w/w) biomass of thermophilic fungi in powder form. Thus, in one embodiment, the present invention provides biomass of no more than 42 (w/w) of thermophilic fungi in cake form, or no more than 13 (w/w) of biomass of thermophilic fungi in powder form. Relevant to foods that are meat substitutes containing biomass. The meat substitute preferably contains at least 1, 2, 5, 10 or 15 (w/w) % biomass from thermophilic fungi in cake form, or at least 0.5, 1, 2 or 5 (w/w) w) % in powder form.

In one embodiment, thermophilic fungal-derived biomass is used as an ingredient for hybrid meat products, where a portion of the meat content is replaced with other protein sources, usually of plant origin. Preferably, the hybrid meat product comprises no more than 52 (w/w)% biomass of thermophilic fungi in cake form, or no more than 16% (w/w)% biomass of thermophilic fungi in powder form. . Therefore, in one embodiment, the present invention provides a biomass of no more than 52 (w/w) of thermophilic fungi in cake form, or no more than 16 (w/w) of biomass of thermophilic fungi in powder form. It relates to foods that are hybrid meat products containing biomass. The hybrid meat product preferably contains at least 1, 2, 5, 10 or 15 (w/w) % biomass from thermophilic fungi in cake form, or at least 0.3, 0.6, 1.5, 3 (w/w)% or 4.5 (w/w)% in powder form.

In one embodiment, the thermophilic fungus-derived biomass is used as an ingredient in the bakery industry, eg as a replacement for or in addition to cereal (or legume) flour. Such cereal and/or legume flours include cereal flours (e.g. wheat, spelt, barley, rye, holly sun, emmer wheat, einkorn, triticale (containing gluten)), cereal flours (e.g. oats, maize). , millet, sorghum, quinoa, rice (gluten-free)), legume flours (eg, chickpea flour, soybean flour), and flours from tubers (eg, potato flour, tapioca flour). An example of a bakery product is bread, but the thermophilic fungus-derived biomass can also be used as an ingredient for other cereal-based food products, such as pasta and similar, premixes (dry) for baked products, biscuits, Cakes, pancakes, porridge (dried/ready to eat), cereal bars, cereal flakes, muesli, bread, gluten-free bread and pizza bases (cooked), including but not limited to. Preferably, the bakery products contain no more than 10% (w/w) of thermophilic fungal biomass in powder form as a replacement for or in addition to the cereal (or legume) flours disclosed herein. Including in addition. Therefore, in one embodiment, the present invention provides 10 (w/w)% thermophilic fungal biomass as a replacement for or in addition to the cereal (or legume) flour disclosed herein. related to bakery products, including Bakery products preferably contain at least 1, 2, 5 or 10 (w/w) of thermophilic fungal-derived biomass in powder form of the cereal (or legume) flour disclosed herein. Including as a replacement or in addition thereto.

Preferably, the cereal-based food product contains no more than 33 (w/w) % thermophilic fungal biomass in cake form, or no more than 10 (w/w) % thermophilic fungal biomass in powder form. include. Therefore, in one embodiment, the present invention provides biomass of no more than 33 (w/w) of thermophilic fungi in cake form, or no more than 10 (w/w) of biomass of thermophilic fungi in powder form. Relevant to foods that are cereal-based foods containing biomass. The cereal-based food preferably contains at least 1, 2, 5, 10 or 15 (w/w) % biomass from thermophilic fungi in cake form, or at least 0.3, 0.6, 1.5 , 3 or 4.5 (w/w)% in powder form.

In one embodiment, thermophilic fungus-derived biomass is used as an ingredient in ready-to-eat meals such as savory pies, pizza-like dishes, savory tarts, soups and porridge. Preferably, the ready-to-eat dietary food product contains no more than 16% (w/w) of biomass of thermophilic fungi in cake form or no more than 5.3% (w/w) of biomass of thermophilic fungi in powder form. Contains fungal biomass. Thus, in one embodiment, the present invention provides 16 (w/w)% or less of thermophilic fungal biomass in cake form, or 15.3 (w/w)% or less of thermophilic fungal biomass in powder form. It relates to foods that are ready-to-eat foods containing fungal biomass. The ready-to-eat dietary food product preferably contains at least 1, 2, 5 or 10 (w/w) % biomass from thermophilic fungi in cake form, or at least 0.5, 1 or 2 (w/w) )% in powder form.

In one embodiment, the thermophilic fungus-derived biomass is used in a soup, preferably ready-to-eat, comprising one or more of water, stock vegetables, meat, fruit, pasta, seasonings, aromas, flavorants and stabilizers. used as an ingredient in soups Preferably, the soup comprises no more than 25% (w/w) of biomass of thermophilic fungi in cake form or no more than 7.5% (w/w) of biomass of thermophilic fungi in powder form. . Thus, in one embodiment, the present invention provides biomass of no more than 25 (w/w)% of thermophilic fungi in cake form, or no more than 7.5% (w/w) of thermophilic fungal biomass in powder form. It relates to food products that are soups, preferably ready-to-eat soups, containing fungal biomass. Soup products preferably contain at least 1, 2, 5, 10 or 15 (w/w) % biomass from thermophilic fungi in cake form, or at least 0.5, 1, 2 or 5 (w/w) )% in powder form.

In one embodiment, the thermophilic fungus-derived biomass is used as an ingredient in food for sports enthusiasts. Foods for sports enthusiasts include, for example, proteins containing one or more of a protein source (e.g. whey protein, soy protein, legume protein), flavoring agents, stabilizers, sugars, coloring agents, and finally minerals and vitamins. It can be a shake or protein bar. Preferably, the food product for sports enthusiasts comprises no more than 70% (w/w) biomass of thermophilic fungi in caked or powder form. Therefore, in one embodiment, the present invention provides a food product for sports enthusiasts, preferably a protein shake or protein shake, comprising no more than 70% (w/w) biomass of thermophilic fungi in caked or powdered form. Related to bars. Foodstuffs for sports enthusiasts preferably contain at least 1, 2, 5, 10, 20 or 40% (w/w) of biomass from thermophilic fungi, either in cake or powder form.

Besides its use in food, the thermophilic fungal biomass of the present invention is a valuable alternative for feed products such as pet food due to its structure (both cake and powder) and nutritional value. material. Therefore, in one embodiment, thermophilic fungal-derived biomass is used as an ingredient in pet food. As used herein, the term "pet" means a domestic or domesticated animal including, but not limited to, domestic dogs, cats, ferrets, rabbits, pigs, and the like. Pet food is herein understood to mean a composition intended for oral consumption that meets one or more nutritional needs of a pet. Pet food may be selected from treats, chews, biscuits, gravies, supplements, toppers, and combinations thereof. Pet food may or may not be nutritionally balanced and complete. Thermophilic fungal biomass represents a good source of dietary fiber, protein and essential amino acids to be used in pet food. The amount of thermophilic fungal biomass in pet food can be prescribed based on the nutritional needs of different pets. Some pets may need to pay more attention to their diet due to weight management or weight loss. In this case, the thermophilic fungal biomass can provide a feeling of satiety to the pet due to its high dietary fiber content. In addition, thermophilic fungal biomass can serve as an alternative protein source for standard animal or vegetable pet food materials. Therefore, in one embodiment, thermophilic fungal biomass is used as a source of protein, amino acids and essential amino acids in pet food, preferably the standard animal or vegetable supply of protein, amino acids in pet food. As an alternative source, it is more preferably used as the sole source of proteins and/or amino acids.

GENERAL DEFINITIONS In this document and its "claims", the verb "comprise" and its conjugations mean that items following the word are included but do not exclude items not specifically recited. is used in its non-limiting sense. In addition, references to an element by the indefinite article "a" or "an" indicate that there may be more than one element present unless the context clearly requires that one and only one element be present. do not rule out the possibility. The indefinite article "a" or "an" thus usually means "at least one." When the word "about" or "approximately" is used in conjunction with a numerical value (e.g. about 10), it is preferred that the value may be greater than or less than 5% of the given value. means As used herein, "subject" means any animal, preferably a mammal, most preferably a human. In preferred embodiments, the subject is non-human.

In the context of the present invention, a decrease or increase in the parameter to be evaluated means a change in value corresponding to that parameter of at least 5%. More preferably, the decrease or increase in its value changes by at least 10%, even more preferably by at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. means. In this latter case, it may be the case that there is no longer a detectable value associated with the parameter.

Throughout this document, when percentages are used to express the amount of substances in a mixture, weight percentages are intended unless otherwise stated or explicitly apparent from the context.

The invention has been described above with respect to a number of exemplary embodiments. Modifications and alternative practices of any part or element are possible, and features from the embodiments may be combined where possible. All citations of literature and patent literature are incorporated herein by reference.

Example 1: Production of Rhizomucor pusillus biomass in cake or powder form For precultivation, Rhizomucor pusillus strain CBS143028 was added to 0.17 g/L KCl, _1.3 g/L _KH2PO4 , _Na2HPO . ₄ 0.4 g/L, citric acid 0.5 gr/L, _MgSO4 . 7 aq 0.7 gr/L, _FeSO4 . 7 aq 0.03 gr/L, _CaCl2 . 2 aq 0.035 gr/L, _ZnSO4 . 7 aq 0.04 gr/L, _MnCl2 . 4aq 0.004, _CuSO4 . 5 aq 0.0005 gr/L, _CoCl2 . 6 _aq 0.0005 gr/L, _Na2B4O7 . 10aq 0.003 gr/L, KI _0.0003 gr/L, _Na2MoO4 . 2 aq 0.0005 gr/L, 11 g dextrose per liter; 4 g ( _NH4 ) _2SO4 per liter; and _7.5 g tartaric acid per liter in 200 ml defined mineral medium at pH 5.5. The preculture was incubated for 24 hours at 46° C. in an orbital shaker at 200 rpm in a 1 L Erlenmeyer flask with a baffled vented stop. This preculture was then used to have a pH of 3.5 and contained 77 g dextrose/L as C source, 1.4 g/L ( _NH4 ) _2SO4 as N source and _NH4 as titrant. A fermenter containing defined mineral medium as described above supplemented with ₃ was inoculated. The fungus was grown in a fermentor in fed-batch mode with a doubling time of 12 hours. Olive oil was continuously fed to maintain a concentration of 50 ppm.

Fermentation broth that reached a dry matter content in the range of 2-5 weight percent was concentrated using vibrating sieves to achieve a minimum of 10% (w/w) dry matter. The biomass is then mixed with antioxidants and pasteurized.

The sieved biomass was then compressed using a hydraulic press to obtain Rhizomucor pcilus biomass as a cake with a dry matter content of about 29 (w/w)%.

A portion of the biomass cake was further freeze-dried and ground (6000 rpm, 1 mm mesh size) to obtain Rhizomucor pcilus biomass in powder form with a dry matter content of about 96 (w/w)%. .

Example 2: Nutritional value of Rhizomucor pusillus biomass Due to its lower moisture content (Table 2), powder from Rhizomucor pusillus biomass concentrates about 3 times higher than cake substitute. The nutritional value of Rhizomucor pusillus biomass is mainly due to its high protein and dietary fiber content (Table 3).

In addition, all nine essential amino acids are contained in Rhizomucor pusillus biomass (Table 4). This makes Rhizomucor pusillus-derived biomass a highly nutritious protein source in the human diet. It must be taken into account that the protein content of Rhizomucor pusillus biomass shown in Table 3 is based on the Kjeldahl method, which is the standard method used to analyze the protein content of different food products. The Kjeldahl method is based on total nitrogen content and uses a standard conversion factor of 6.25 to estimate the protein content in the foods analyzed. However, a conversion factor of 6.25 may not be suitable for certain foods. In Rhizomucor pusillus biomass, the conversion factor may be overestimated due to the presence of other nitrogen sources such as RNA, chitin and chitosan. The true protein content of Rhizomucor pusillus biomass can be better estimated through amino acid analysis for this reason. Among amino acids, Rhizomucor pusillus biomass has a particularly high content of citrulline (Table 4).

The dietary fiber content on a dry matter basis of Rhizomucor pusillus biomass is extremely high when compared to other foods known to be good sources of dietary fiber (Table 5). The dietary fiber content of Rhizomucor pusillus biomass is even higher (up to 95%) than that of mycoprotein, the main source of Quorn brand meat substitutes.

Table 6 shows the content of chitin and chitosan in various batches of Rhizomucor pusillus biomass. Interestingly, the Rhizomucor pusillus biomass not only contains chitin as a fiber, but also an almost equal amount of fiber chitosan. Chitosan is a deacetylated form of chitin and health benefits have been described for chitosan in animals. It is also anticipated that human chitosan will also prove to be beneficial to health [16]. As much as 9.2% (w/w) of the Rhizomucor pusillus biomass is chitosan. This is in contrast to the mycoprotein source Fusarium venenatum, whose dietary fiber consists of one-third chitin and two-thirds beta-glucan, according to Finnigan et al. , 6) [17].

Example 3: Nutritional Value of Meat Substitute Made with Rhizomucor Psillus Biomass Prepared with 32.3% textured plant protein extrudate, 1.3% tex fiber and chicken flavor (mix ingredients, form and roast in cooking oven at 185°C for 35 seconds). at 170° C. for 8 minutes and freeze until a temperature of −20° C. is reached in the center). Compared to the vegetarian hamburger, the protein content (based on Kjeldahl analysis) of the meat substitute made with Rhizomucor pcilus biomass is slightly lower (Table 7). The meat substitute with Rhizomucor pusillus biomass evaluated was made with a biomass cake content of only 28% (w/w), which was set for Rhizomucor pusillus biomass when used as a meat substitute. It must be considered much lower than the maximum level (42% w/w). This indicates that the protein and dietary fiber content of meat substitutes made with Rhizomucor pusillus biomass will vary according to the percentage of Rhizomucor pusillus biomass in the final product and other ingredients mixed with Rhizomucor pusillus biomass. , means that it can be modified.

Example 4 Micronutrient Content of Rhizomucor Psillus Biomass Rhizomucor pusillus biomass was analyzed for micronutrients and various micronutrients including different vitamins such as vitamins A, E, and B group vitamins, as well as choline. (see Table 8).

Rhizomucor pusillus biomass has a particularly high content of choline (Table 8). Choline was once also called vitamin B4 [6] [7]. Choline has important functions in the structural integrity of cell membranes and in different metabolic pathways in the human body such as methyl metabolism, cholinergic neurotransmission, transmembrane signaling, lipid and cholesterol transport and metabolism [8 ]. Choline can be synthesized de novo by the human body. However, choline still represents a fundamental component of the human diet because the amount de novo produced is 400 mg/day in adults (both genders) Adequate Intake (AI) ) [9]. In 1998, choline was recognized as an essential nutrient by the Institute of Medicine (IOM) [8][10]. Table 9 shows a comparison of the choline content of Rhizomucor pusillus biomass with the choline content of several types of food.

The vitamin A content of Rhizomucor pusillus biomass cake is 0.3 mg/kg (Table 8). However, vitamin A is also believed to come from materials used during biomass production, such as olive oil or antioxidants. However, the vitamin A content of Rhizomucor pcilus biomass produced without the use of olive oil and antioxidants was still 0.1 mg/kg, with at least part of the vitamin A produced by the fungus during the fermentation process. It is suggested that

The same thing happens with vitamin E. Vitamin E occurs naturally in olive oil (approximately 5.1 mg/100 g according to the NEVO table) and appears to be present in antioxidants. The vitamin E (dl-alpha tocopherol acetate) content of Rhizomucor pusillus biomass cake is 2.5 mg/kg (Table 8). However, when the Rhizomucor pusillus biomass cake was produced without the use of olive oil and without the addition of antioxidants, it still had a vitamin E content of 22 mg/kg, indicating that vitamin E is absorbed by fungi during the fermentation process. suggesting that it is produced. In this regard, it is interesting to note that the vitamin E content of fungal biomass made without olive oil and antioxidants is even higher than in biomass produced in their presence.

In addition, Table 8 shows that Rhizomucor pusillus biomass also contains many vitamins of the B group, in particular vitamin B2 (riboflavin), vitamin B6 (pyridoxine) and folic acid (vitamin B11, some of which are French, German and American). Also known as vitamin B9 in the country).

Example 5: Mineral Content of Rhizomucor Psillus Biomass As shown in Table 10, Rhizomucor pusillus biomass contains high amounts of different minerals added during the fermentation process and necessary for fungal growth. The iron (Fe) content in Rhizomucor pusillus biomass was particularly high and was the limiting factor for intake assessment according to EFSA DRV. The iron dosage for Rhizomucor pusillus biomass production should be reduced to reach a final concentration of 14 mg/kg (Table 10). Additionally, zinc was also reduced to a final concentration of 16 mg/kg (Table 10).

Table 11 presents a comparison of the iron content of Rhizomucor pusillus biomass with that of various other types of food. It should be recognized that although some of the meat products in Table 11 contain lower amounts of iron, their bioavailability is higher because the iron is present in the heme-bound form.

Example 6 Lipid Content of Rhizomucor pusillus Biomass As shown in Table 3 above, Rhizomucor pusillus biomass contains about 9% (w/w) on a dry matter basis. A more detailed analysis showed that, on average, 22.2 (w/w)% consisted of saturated fatty acids, 50.1 (w/w)% consisted of monounsaturated fatty acids (mostly oleic acid), and 27.7%. (w/w) consisted of polyunsaturated fatty acids (mostly linoleic acid). Rhizomucor pusillus biomass is thus relatively rich in monounsaturated and polyunsaturated fatty acids.

References
[1] EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion on Dietary Reference Values for carbohydrates and dietary fiber," 2010.
[2] P. Sharma, C.Bhandari, S. Kumar, B. Sharma, P. Bhadwal and N. Agnihotri, "Chapter 11 -Dietary Fibers: A Way to a Healthy Microbiome," in Diet, Microbiome andHealth, Handbook of Food Bioengineering, 2018, pp. 299-345.
[3] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), "Scientific Opinion on Dietary Reference Values for protein," 2012.
[4] T. Suzuki, M. Morita, Y. Kobayashi and A. Kamimura, "Oral L-citrulline supplementation enhancements cycling time trial performance in healthy trained men: Double-blindrandomized placebo-controlled 2-way crossover study.," Journal of the International Society of Sports Nutrition, vol. 13, no. 6, 2016.
[5] FEDIAF(European Pet Food Industry), "Nutritional needs for cats and dogs," 2018.
[6] T. Navarra, The Encyclopedia of Vitamins, Minerals, and Supplements., Infobase Publishing, 2004.
[7] F. Macdonaldand RL Lundblad, Handbook of Biochemistry and Molecular Biology, Fourthedition, CRC Press, 2010.
[8] Institute of Medicine (US) Standing Committee on the Scientific Evaluation of Dietary Reference Intakes and its Panel on Folate, Other B Vitamins ancholine,"Dietary Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B6, Folate, Vitamin B12, Pantothenic Acid , Biotin, and Choline," 1998.
[9] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), "Dietary Reference Values for choline," 2016.
[10] SH Zeiseland KA Da Costa, "Choline: An Essential Nutrient for PublicHealth," Nutrition Reviews, vol. 67, pp. 615-623, 2009.
[11] FEDIAF(European Pet Food Industry), "Nutritional Guidelines For Complete and Complementary Pet Food for Cats and Dogs," 2018.
[12] EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion on Dietary Reference Values for vitamin A," 2015.
[13] EFSA Panel on Dietetic Products, Nutrition, and Allergies (NDA), "Scientific Opinion on Dietary Reference Values for vitamin E as α-tocopherol," 2015.
[14] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), "Dietary Reference Values for riboflavin," 2017.
[15] EFSA Panel on Dietetic Products, Nutrition and Allergies (NDA), "Dietary Reference Values for vitamin B6," 2016.

Claims (15)

of a composition comprising thermophilic fungal biomass as a source of at least one of a) proteins and/or amino acids, b) lipids, c) dietary fiber, d) choline and/or vitamins and e) minerals, Use in at least one of food, beverage, pet food and feed products. The composition comprising thermophilic fungal biomass is removed from the medium by: a) growing a strain of thermophilic fungus in submerged culture in a medium; recovering said fungal biomass from the sieved, filtered or decanted biomass cake preferably with a dry matter concentration of at least 12% (w/v) and c) optionally further drying said biomass cake by pressing out remaining water to obtain a biomass cake having a dry matter concentration of at least 20%. 2. Use according to claim 1, which is a cake that can be prepared. Said composition comprising thermophilic fungal biomass is obtained by grinding the biomass cake obtained in claim 2, by warm air, by freeze-drying, preferably under vacuum or by flash-drying, preferably 7(w /w)% or less to a biomass powder to a dry powder obtainable by further drying to a moisture content. The fungal strains are Rasamsonia, Talaromyces, Penicillium, Acremonium, Humicola, Paecilomyces, Chaetomium, Rhizomucor mucor), Rhizopus , Thermomyces, Myceliophthora, Thermoascus, Thielavia, Mucor, Stibella, Melanocarpus, Malbranchea, Dactylo Dactylomyces, Canary myces a strain of the fungal genus selected from the group consisting of Canariomyces, Scytalidium, Myriococcum, Corynascus and Coonemeria, more preferably said genus is Rhizomucor, and Preferably, said strain is Rhizomucor pusillus, most preferably said strain is Rhizomucor pusillus CBS143028, or a strain that is a single colony isolate or derivative thereof. 4. Use according to any one of 1 to 3. at least one of the proteins and amino acids is aspartic acid, asparagine, threonine, serine, glutamic acid, glutamine, proline, glycine, alanine, citrulline, valine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, arginine, cysteine, An amino acid selected from the group consisting of methionine and tryptophan, preferably said amino acid is an essential amino acid, more preferably from the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine and tryptophan. Use according to any one of claims 1 to 4, which is a selected essential amino acid, most preferably in vegetarian and vegan foods. a) said biomass of thermophilic fungi is used as a (dietary) source of at least one of proteins, amino acids and essential amino acids in vegetarian or vegan foods,
b) said biomass of thermophilic fungi is used as a (dietary) source of citrulline in food, food supplements or beverages for sports enthusiasts,
c) said biomass of thermophilic fungi as a source of protein, amino acids and essential amino acids in pet food, preferably as a standard animal or vegetable source of protein, amino acids in pet food; Use according to claim 5, used as a replacement, more preferably as the sole source of proteins and/or amino acids. wherein said biomass of said thermophilic fungus is used in: i) meat substitutes, ii) hybrid foods containing meat and non-meat ingredients, iii) carbohydrate rich foods, iv) bakery products made with refined flours and v) pet food, used as dietary fiber source to enrich the fiber content of food products selected from, preferably the dietary fiber comprises chitosan. Use as described. Said biomass of said thermophilic fungus is used as a source of dietary fiber in food for humans or pets, preferably for providing a feeling of satiety to humans or pets in need of weight control or weight loss, preferably said food Use according to claim 7, wherein the fibers comprise chitosan. Claims 1 to 8, wherein the biomass of the thermophilic fungus is used as a choline source in vegan or vegetarian food or in pet food, preferably cat or dog food. Use of. Said biomass of said thermophilic fungus is used as a source of at least one of retinol, vitamin E, riboflavin, pyridoxine and folic acid, preferably said biomass is used as a source of retinol in cat food. , the use according to any one of claims 1-9. Said biomass of said thermophilic fungus is used as iron source, preferably said biomass of said thermophilic fungus is used to replace soybean as iron source, more preferably said biomass of said thermophilic fungus is used to replace soybean as iron source in meat substitutes. The biomass of the thermophilic fungus is used as a source of at least one of monounsaturated and polyunsaturated fatty acids, preferably the biomass thermophilic fungus contains at least one of oleic acid and linoleic acid. Use according to any one of claims 1 to 11, used as one source. said biomass of said thermophilic fungus in either cake or powder form in food, beverages, pet food and feed products at a maximum level of 70% (w/w) and at least 1 (w/w); Use according to any one of claims 1 to 12, used for incorporation at a minimum level of %. said biomass of said thermophilic fungus comprising:
a) a meat substitute, wherein no more than 42 (w/w) % of said biomass of said thermophilic fungus in cake form or no more than 14 (w/w) % of said thermophilic fungus in powder form a meat substitute comprising said biomass of
b) a hybrid meat product comprising no more than 52 (w/w)% of said biomass of said thermophilic fungus in cake form, or no more than 17.3% (w/w) of said biomass in powder form; a hybrid meat product comprising said biomass of thermophilic fungi;
c) a cereal-based food product, wherein no more than 30% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 10% (w/w) of said thermophilic fungus in powder form a cereal-based food product comprising said biomass of fungi;
d) a ready-to-eat dietary food product comprising no more than 16% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 5.3% (w/w) in powder form a ready-to-eat dietary food product comprising said biomass of said thermophilic fungus;
e) a ready-to-eat soup comprising no more than 27% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 9% (w/w) of said thermophilic fungus in powder form f) a food product for sports enthusiasts, comprising no more than 70% (w/w) of said biomass of said thermophilic fungus in cake form or in powder form. Use according to any one of claims 1 to 13, used as an ingredient for food for sports enthusiasts, comprising A food comprising a composition comprising the biomass of the thermophilic fungus according to any one of claims 1 to 4,
a) a meat substitute, 42 (w/w)% or less of said biomass of said thermophilic fungus in cake form, or 14 (w/w)% or less of said thermophilic fungus in powder form; a meat substitute comprising said biomass;
b) being a hybrid meat product, no more than 52% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 17.3% (w/w) of said thermophilic fungus in powder form; a hybrid meat product comprising said biomass of fungi;
c) a cereal-based food product with no more than 30% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 10% (w/w) of said thermophilic fungus in powder form. a cereal-based food product comprising said biomass of
d) It is a ready-to-eat dietary food product and contains no more than 16% (w/w) of said biomass of said thermophilic fungus in cake form or no more than 5.3% (w/w) of said biomass in powder form. a ready-to-eat dietary food product comprising said biomass of said thermophilic fungus;
e) a ready-to-eat soup, no more than 27% (w/w) of said biomass of said thermophilic fungus in cake form, or no more than 9% (w/w) of said thermophilic fungus in powder form. and f) a food product for sports enthusiasts, comprising no more than 70% (w/w) of said biomass of said thermophilic fungus in cake form or in powder form. , food that is food for sports enthusiasts.